Mitochondria comprise approximately 30% of the total intracellular volume of a mammalian cardiomyocyte. Not surprisingly, subtle alterations in mitochondrial function or membrane potential can have a dramatic influence on cardiomyocyte energy production and ultimately, the life or death individual cell. Indeed, cellular injury or stress stimulation directly elicit alterations in mitochondrial architecture, membrane potential, and oxidative capacity, which can be associated with ah irreversible loss of mitochondrial matrix contents and integral membrane protein constituents such as cytochrome C oxidase, followed soon thereafter by activation of intracellular proteases and DNA fragmentation enzymes associated with apoptosis and necrosis. An emerging paradigm places mitochondrial permeability transition pore (MPTP) formation as a central event precipitating cardiac myocyte apoptosis or necrosis following ischemic injury and during progressive heart failure. This project will test the hypothesis that MPTP is a primary mechanism responsible for driving myocardial cell death following ischemia- reperfusion injury or in response to long-standing cardiomyopathy. Both gain- and loss-of-function approaches will be implemented in the mouse as a means of dissecting the molecular determinants of MPTP and potential therapeutic opportunities. Specific aim 1 will define the mechanism of MPTP formation and its functional consequence in regulating cardiac myocyte apoptosis. Specific aim 2 will determine if inducible MPTP formation regulates cardiac myocyte apoptosis in vivo, while Specific aim 3 will target VDAC1/3 and cyclophilin D as a means of blocking MPTP formation in the heart. Even though inhibition of MPTP formation with pharmacologic agents often prevents cell death following catastrophic stimuli in diverse cell-types, the necessity of MPTP formation in mediating cardiomyocyte cell death following ischemic injury or long-standing cardiomyopathy has not been elucidated. Moreover, the identity and key functions of the putative MPTP components have not been subjected to genetic gain- or loss-of-function analysis in vivo, leaving the true identity of the complex unresolved. A greater understanding of the key constituents that comprise the MPTP, as well as further characterizing its functional dominance in the heart, will likely suggest novel approaches for treating human heart disease associated with cell death. [unreadable] [unreadable] [unreadable]